Peroxidase variants with improved hydrogen peroxide stability

ABSTRACT

A variant of a Coprinus cinereus peroxidase having one or more substitutions selected from M166L, V, I, F, Q and M242L, V, I, F, Q, wherein the parent peroxidase is encoded by the DNA sequence shown of SEQ ID:1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/331,515filed on Nov. 1, 1994 now U.S. Pat. No. 5,851,811, which is acontinuation of application Ser. No. PCT/DK93/00189 filed on Jun. 1,1993, and claims priority under 35 U.S.C. 119 of Danish applicationserial no. 0729/92 filed Jun. 1, 1992, the contents of which are fullyincorporated herein by reference.

FIELD OF INVENTION

The present invention relates to a novel variant of peroxidase, and ableaching agent or detergent composition comprising the peroxidasevariant.

BACKGROUND OF THE INVENTION

The use of bleaching agents in washing procedures and as constituents ofdetergent compositions is well known in the art. Thus, bleaching agentsare incorporated in or sold as constituents of a major part of thecommercially available detergent compositions. Important conventionalbleaching agents incorporated in detergent compositions are compoundswhich act as precursors of hydrogen peroxide formed in the course of thewashing procedure. Perborates and percarbonates are the most importantexamples of compounds which are employed as bleaching agents and whichexert a bleaching effect in this fashion. The detailed mechanism ofbleaching by means of these bleaching agents is not known at present,but it is generally assumed that the hydrogen peroxide formed duringwashing converts coloured substances (responsible for stains on fabric)into non-coloured materials by oxidation and that some oxidation of thecoloured substances may also take place due to their direct interactionwith perborate or percarbonate.

One drawback of these commonly used bleaching agents is that they arenot particularly efficient at the lower temperatures at which colouredfabrics are usually washed. Their efficiency may be enhanced by the useof activators (e.g. organic acid anhydrides, esters or imides) whichgive rise to the formation of peracids.

Apart from being employed for bleaching stains on fabric, suchconventional bleaching agents have also been suggested for preventingsurplus dyes from coloured fabrics which leach from the fabrics whenthese are washed from being deposited on other fabrics present in thesame wash (this phenomenon is commonly known as dye transfer). Theproblem of dye transfer, of course, is most noticeable when white orlight-coloured fabrics are washed together with fabrics of a darkercolour from which dye is leached during washing.

It has been found that peroxidases utilizing hydrogen peroxide as theirsubstrate are able to enhance the bleaching effect of hydrogen peroxideduring washing. The use of peroxidase for bleaching stains on fabrics isdescribed in WO 89/09813. It was also found that coloured substancesleached from dyed fabrics could be bleached by means of peroxidases. Theuse of peroxidase for inhibiting the transfer of dye from a dyed fabricto another fabric during washing is described in WO 91/05839.

SUMMARY OF THE INVENTION

It has surprisingly been found that peroxidase variants with an improvedstability towards hydrogen peroxide may be prepared by recombinant DNAtechniques.

Accordingly, the present invention relates to a peroxidase variant withimproved hydrogen peroxide stability, characterized by insertion,deletion or substitution of one or more amino acid residues located inor near the substrate channel of the parent peroxidase, near the hemegroup of the parent peroxidase or at or near the active site of theparent peroxidase.

Information about the three-dimensional structure of the parentperoxidase was obtained by aligning the amino acid sequence of theparent peroxidase to amino acid sequences of other known peroxidases (K.G. Welinder et al., “Structure and evolution of peroxidases” in PlantPeroxidase Biochemistry and Physiology, K. G. Welinder et al. (eds.),University of Copenhagen and Geneva 1993, in press). The sequencealignment showed that the parent peroxidase was homologous to yeastcytochrome c peroxidase (CCP) which has a known structure (J. Wang etal. , Biochemistry 29, 1990, p. 7160), and the overall structure of theparent peroxidase could therefore be inferred from the CCP structure. Anexample of how structural information for a homologous peroxidase can beinferred from the crystal structure of CCP is given in P. Du et al.,Protein Engineering 5, 1992, pp. 679-691.

In the present context, the term “substrate channel” is intended toindicate a passage in the peroxidase molecule through which thesubstrate passes before oxidation at the heme group of the peroxidase.The term “active site” is intended to indicate the substrate-bindingsite of the peroxidase (cf. Eur. J. Biochem. 213, 1993, pp 605-611). Theterm “near” should be taken to indicate a distance from the substratechannel, heme group or active site, respectively, of not more than 15 Å,more preferably not more than 10 Å, most preferably not more than 5Å, inany direction. The term “stability” is intended to indicate that theenzyme is active in the presence of hydrogen peroxide at a concentrationof up to 20 mM.

In another aspect, the present invention relates to a bleaching agentcomprising a peroxidase variant according to the invention, optionallyin the form of a non-dusting granulate, a liquid, in particular astabilised liquid, or a protected enzyme.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic representation of the plasmid pHD414.

DETAILED DISCLOSURE OF THE INVENTION

In the present description and claims, the following abbreviations areused:

A = Ala = Alanine V = Val = Valine L = Leu = Leucine I = Ile =Isoleucine P = Pro = Proline F = Phe = Phenylalanine W = Trp =Tryptophan M = Met = Methionine G = Gly = Glycine S = Ser = Serine T =Thr = Threonine C = Cys = Cysteine Y = Tyr = Tyrosine N = Asn =Asparagine Q = Gln = Glutamine D = Asp = Aspartic Acid E = Glu =Glutamic Acid K = Lys = Lysine R = Arg = Arginine H = His = Histidine

In describing peroxidase variants according to the invention, thefollowing nomenclature is used for ease of reference: Original aminoacid(s):position(s):substituted amino acid(s)

According to this nomenclature, for instance the substitution ofglutamic acid for glycine in position 154 is shown as:

G154E

a deletion of glycine in the same position is shown as:

G154*

and insertion of an additional amino acid residue such as lysine isshown as:

G154GK

Multiple mutations are separated by pluses, i.e.:

G154E+G156F

representing mutations in positions 154 and 156 substituting glutamicacid and phenylalanine for glycine and glycine, respectively.

The parent peroxidase may suitably be a microbial, in particular afungal, peroxidase, preferably a Coprinus sp. or Arthromyces sp.peroxidase, in particular a Coprinus cinereus peroxidase.

In a preferred embodiment of the peroxidase variant according to theinvention, the parent peroxidase is encoded by the DNA sequence shown inSEQ ID No. 1. Said sequence is derivable from Coprinus cinereus.

In one embodiment of the peroxidase variant of the invention, one ormore amino acid residues are deleted, inserted or substituted in theregion from amino acid residue 79 to 94, 125, 153 to 157, 161 to 204,242, 276 or 279 of the parent peroxidase encoded by the DNA sequenceshown in SEQ ID No. 1.

In other embodiments of the peroxidase variant according to theinvention, one or more amino acid residues may suitably be substitutedas follows

Q1S,E,

S8N,

C22S,

C23S,

Q38N,

D56N,T,I,

M125L,V,I,F,Q,

N142S,T,P,D,

G154E,

G156F,

N157E,F,

M166L,V,I,F,Q,

M242L,V,I,F,Q,

D245I,T,N,

C256S,

S263N,

N265S,

M276I,F,L,V,Q,

M279L,V,I,F,Q,

A304S,

T331A,N,

S338A

In an alternative embodiment, the peroxidase variant according to theinvention may be a fragment of the parent peroxidase, e.g. a fragmentfrom amino acid residue 1 to amino acid residue 304 of the peroxidasesequence encoded by the DNA sequence shown in SEQ ID No. 1.

According to the invention, two or more amino acid residues of theperoxidase sequence may also be substituted as follows

G154E+G156F+N157E

G154E+G156F

G156F+N157F

T331A+S338A

S263N+N265S

T331N+S338A

T331N+S338A+N142

T331N+S338A+S263N+N265S+S8N

T331N+S338A+S263N+N265S+S8N+Q38N+A304S

The DNA sequence encoding a parent peroxidase may be isolated from anymicroorganism producing the peroxidase in question by various methodswell known in the art. First a genomic DNA and/or cDNA library should beconstructed using chromosomal DNA or messenger RNA from the organismthat produces the peroxidase to be studied. Then, if the amino acidsequence of the peroxidase is known, homologous, labelledoligonucleotide probes may be synthesized and used to identifyperoxidase-encoding clones from a genomic library of bacterial DNA, orfrom a fungal cDNA library. Alternatively, a labelled oligonucleotideprobe containing sequences homologous to peroxidase from another strainof fungus could be used as a probe to identify peroxidase-encodingclones, using hybridization and washing conditions of lower stringency.

Another method for identifying peroxidase-producing clones involvesinserting fragments of genomic DNA into an expression vector, such as aplasmid, transforming peroxidase-negative bacteria with the resultinggenomic DNA library, and then plating the transformed bacteria onto agarcontaining a substrate for peroxidase. Those bacteria containingperoxidase-bearing plasmid will produce colonies surrounded by a halo ofclear agar, due to digestion of the substrate by secreted peroxidase.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g. the phosphoamiditemethod described by S. L. Beaucage and M. H. Caruthers, TetrahedronLetters 22, 1981, pp. 1859-1869, or the method described by Matthes etal., The EMBO J. 3, 1984, pp. 801-805. According to the phosphoamiditemethod, oligonucleotides are synthesized, e.g. in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in appropriatevectors.

Finally, the DNA sequence may be of mixed genomic and synthetic, mixedsynthetic and cDNA or mixed genomic and cDNA origin prepared by ligatingfragments of synthetic, genomic or cDNA origin (as appropriate), thefragments corresponding to various parts of the entire DNA sequence, inaccordance with standard techniques. The DNA sequence may also beprepared by polymerase chain reaction (PCR) using specific primers, forinstance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et al.,Science 239, 1988, pp. 487-491.

Once a peroxidase-encoding DNA sequence has been isolated, and desirablesites for mutation identified, mutations may be introduced usingsynthetic oligonucleotides. These oligonucleotides contain nucleotidesequences flanking the desired mutation sites; mutant nucleotides areinserted during oligonucleotide synthesis. In a specific method, asingle-stranded gap of DNA, bridging the peroxidase-encoding sequence,is created in a vector carrying the peroxidase gene. Then the syntheticnucleotide, bearing the desired mutation, is annealed to a homologousportion of the single-stranded DNA. The remaining gap is then filled inwith DNA polymerase I (Klenow fragment) and the construct is ligatedusing T4 ligase. A specific example of this method is described inMorinaga et al., (1984, Biotechnology 2:646-639). U.S. Pat. No.4,760,025, by Estell et al., issued Jul. 26, 1988, discloses theintroduction of oligonucleotides encoding multiple mutations byperforming minor alterations of the cassette, however, an even greatervariety of mutations can be introduced at any one time by the Morinagamethod, because a multitude of oligonucleotides, of various lengths, canbe introduced.

Another method of introducing mutations into peroxidase-encodingsequences is described in Nelson and Long, Analytical Biochemistry 180,1989, pp. 147-151. It involves the 3-step generation of a PCR fragmentcontaining the desired mutation introduced by using a chemicallysynthesized DNA strand as one of the primers in the PCR reactions. Fromthe PCR-generated fragment, a DNA fragment carrying the mutation may beisolated by cleavage with restriction endonucleases and reinserted intoan expression plasmid.

According to the invention, a mutated peroxidase-coding sequenceproduced by one of the methods described above, or any alternativemethods known in the art, can be expressed, in enzyme form, using anexpression vector which typically includes control sequences encoding apromoter, operator, ribosome binding site, translation initiationsignal, and, optionally, a repressor gene or various activator genes. Topermit the secretion of the expressed protein, nucleotides encoding asignal sequence may be inserted prior to the peroxidase-coding sequence.For expression under the direction of control sequences, a target geneis operably linked to the control sequences in the proper reading frame.Promoter sequences that can be incorporated into plasmid vectors, andwhich can support the transcription of the mutant peroxidase gene,include but are not limited to the prokaryotic β-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731) and the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Further references can also be found in“Useful proteins from recombinant bacteria” in Scientific American,1980, 242:74-94.

According to one embodiment B. subtilis is transformed by an expressionvector carrying the mutated DNA. If expression is to take place in asecreting microorganism such as B. subtilis a signal sequence may followthe translation initiation signal and precede the DNA sequence ofinterest. The signal sequence acts to transport the expression productto the cell wall where it is cleaved from the product upon secretion.The term “control sequences” as defined above is intended to include asignal sequence, when present.

The host organism transformed with the DNA sequence encoding theperoxidase variant of the invention may also be a yeast, preferably astrain of Saccharomyces, e.g. Saccharomyces cerevisiae orSchizosaccharomyces pombe, or Pichia, e.g. Pichia pastoris.

In a currently preferred method of producing the peroxidase variant ofthe invention, a filamentous fungus is used as the host organism. Thefilamentous fungus host organism may conveniently be one which haspreviously been used as a host for producing recombinant proteins, e.g.a strain of Aspergillus sp., such as A. niger, A. nidulans or A. oryzae.The use of A. oryzae in the production of recombinant proteins isextensively described in, e.g. EP 238 023.

For expression of peroxidase variants in Aspergillus, the DNA sequencecoding for the peroxidase variant is preceded by a promoter. Thepromoter may be any DNA sequence exhibiting a strong transcriptionalactivity in Aspergillus and may be derived from a gene encoding anextracellular or intracellular protein such as an amylase, aglucoamylase, a protease, a lipase, a peroxidase, a cellulase or aglycolytic enzyme.

Examples of suitable promoters are those derived from the gene encodingA. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. nigerneutral α-amylase, A. niger acid stable α-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease orA. oryzae triose phosphate isomerase.

In particular when the host organism is A. oryzae, a preferred promoterfor use in the process of the present invention is the A. oryzae TAKAamylase promoter as it exhibits a strong transcriptional activity in A.oryzae. The sequence of the TAKA amylase promoter appears from EP 238023.

Termination and polyadenylation sequences may suitably be derived fromthe same sources as the promoter.

The techniques used to transform a fungal host cell may suitably be asdescribed in EP 238 023.

To ensure secretion of the peroxidase variant from the host cell, theDNA sequence encoding the peroxidase variant may be preceded by a signalsequence which may be a naturally occurring signal sequence or afunctional part thereof or a synthetic sequence providing secretion ofthe protein from the cell. In particular, the signal sequence may bederived from a gene encoding an Aspergillus sp. amylase or glucoamylase,a gene encoding a Rhizomucor miehei lipase or protease, or a geneencoding a Humicola cellulase, xylanase or lipase. The signal sequenceis preferably derived from the gene encoding A. oryzae TAKA amylase, A.niger neutral α-amylase, A. niger acid-stable α-amylase, Coprinuscinereus or macrorhizus peroxidase, or A. niger glucoamylase.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing Aspergillus cells. Thetransformants are usually stable and may be cultured in the absence ofselection pressure. However, if the transformants are found to beunstable, a selection marker introduced into the cells may be used forselection.

The mature peroxidase protein secreted from the host cells mayconveniently be recovered from the culture medium by well-knownprocedures including separating the cells from the medium bycentrifugation or filtration, and precipitating proteinaceous componentsof the medium by means of a salt such as ammonium sulphate, followed bychromatographic procedures such as ion exchange chromatography, affinitychromatography, or the like.

To obtain an optimal bleaching effect of the peroxidase variant,hydrogen peroxide or a precursor of hydrogen peroxide, preferablyperborate or percarbonate, may advantageously be added to the bleachingagent of the invention as the substrate for the peroxidase variant.

While the mechanism governing the ability of peroxidase to effectbleaching of coloured substances present on fabrics or in solution hasnot yet been elucidated, it is currently believed that the enzyme actsby reducing hydrogen peroxide or molecular oxygen and oxidizing thecoloured substance (donor substrate) dissolved or dispersed in the washliquor, thereby either generating a colourless substance or providing asubstance which is not adsorbed to the fabric. This reaction is shown inReaction Scheme 1 below

Reaction Scheme 1:

By using a peroxidase variant according to the invention which is lesssensitive to hydrogen peroxide, it may be possible to add a smalleramount of the enzyme to the bleaching/washing liquor and yet obtain asatisfactory bleaching effect.

In the bleaching agent, the amount of hydrogen peroxide or hydrogenperoxide precursor preferably corresponds to a hydrogen peroxideconcentration in the wash liquor of between 10 μM and 20 mM.

For use of the present peroxidase variant as a bleaching agent, it hassurprisingly been found that the addition of another oxidisablesubstrate (for the peroxidase variant of the invention) at the beginningor during the washing and/or rinsing process may enhance the bleachingeffect of the peroxidase variant employed. This is thought to beascribable to the formation of short-lived radicals or other oxidisedstates of this substrate which participate in the bleaching or othermodification of the coloured substance. Examples of such oxidisablesubstrates are organic compounds such as phenolic compounds, e.g.7-hydroxycoumarin, vanillin, p-hydroxycinnamic acid, p-hydroxybenzenesulphonate or 2,4-dichlorophenol. Other examples of phenolic compoundswhich may be used for the present purpose are those given in M. Kato andS. Shimizu, Plant Cell Physiol. 26(7), 1985, pp. 1291-1301 (cf. Table 1in particular) or B. C. Saunders et al., Peroxidase, London, 1964, p.141 ff. The amount of oxidisable substrate to be added is suitablybetween about 1 μM and 1 mM.

According to the invention, the peroxidase variant may typically beadded as a component of a detergent composition. As such, it may beincluded in the detergent composition in the form of a non-dustinggranulate, a liquid, in particular a stabilized liquid, or a protectedenzyme. Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) andmay optionally be coated by methods known in the art. Liquid enzymepreparations may, for instance, be stabilized by adding a polyol such aspropylene glycol, a sugar or sugar alcohol, lactic acid or boric acidaccording to established methods. Other enzyme stabilizers are wellknown in the art. Protected enzymes may be prepared according to themethod disclosed in EP 238,216. The detergent composition may alsocomprise one or more substrates for the enzyme.

The detergent composition of the invention may be in any convenientform, e.g. as powder, granules or liquid. A liquid detergent may beaqueous, typically containing up to 70% water and 0-20% organic solvent.

The detergent composition comprises a surfactant which may be anionic,non-ionic, cationic, amphoteric or a mixture of these types. Thedetergent will usually contain 5-30% anionic surfactant such as linearalkyl benzene sulphonate (LAS), alpha-olefin sulphonate (AOS), alkylsulphate (AS), alcohol ethoxy sulphate (AES) or soap. It may alsocontain 3-20% non-ionic surfactant such as nonyl phenol ethoxylate oralcohol ethoxylate.

The detergent composition may additionally comprise one or more otherenzymes, such as an amylase, lipase, cellulase or protease.

The pH (measured in aqueous detergent solution) will usually be neutralor alkaline, e.g. 7-11. The detergent may contain 1-40% of a detergentbuilder such as zeolite, phosphate, phosphonate, citrate, NTA, EDTA orDTPA, alkenyl succinic anhydride, or silicate, or it may be unbuilt(i.e. essentially free of a detergent builder). It may also containother conventional detergent ingredients, e.g. fabric conditioners, foamboosters, anti-corrosion agents, soil-suspending agents, sequesteringagents, anti-soil redeposition agents, stabilizing agents for theenzyme(s), foam depressors, dyes, bactericides, optical brighteners orperfumes.

Particular forms of detergent composition within the scope of theinvention include:

a) A detergent composition formulated as a detergent powder containingphosphate builder, anionic surfactant, nonionic surfactant, silicate,alkali to adjust to desired pH in use, and neutral inorganic salt.

b) A detergent composition formulated as a detergent powder containingzeolite builder, anionic surfactant, nonionic surfactant, acrylic orequivalent polymer, silicate, alkali to adjust to desired pH in use, andneutral inorganic salt.

c) A detergent composition formulated as an aqueous detergent liquidcomprising anionic surfactant, nonionic surfactant, humectant, organicacid, alkali, with a pH in use adjusted to a value between 7 and 10.5.

d) A detergent composition formulated as a non-aqueous detergent liquidcomprising a liquid nonionic surfactant consisting essentially of linearalkoxylated primary alcohol, phosphate builder, alkali, with a pH in useadjusted to a value between about 7 and 10.5.

e) A detergent composition formulated as a detergent powder in the formof a granulate having a bulk density of at least 600 g/l, containinganionic surfactant and nonionic surfactant, low or substantially zeroneutral inorganic salt, phosphate builder, and sodium silicate.

f) A detergent composition formulated as a detergent powder in the formof a granulate having a bulk density of at least 600 g/l, containinganionic surfactant and nonionic surfactant, low or substantially zeroneutral inorganic salt, zeolite builder, and sodium silicate.

g) A detergent composition formulated as a detergent powder containinganionic surfactant, nonionic surfactant, acrylic polymer, fatty acidsoap, sodium carbonate, sodium sulphate, clay particles, and sodiumsilicate.

h) A liquid compact detergent comprising 5-65% by weight of surfactant,0-50% by weight of builder, and 0-30% by weight of electrolyte.

Apart from these ingredients, the detergent compositions a)-h) include aperoxidase variant of the invention and a substrate therefor, andoptionally one or more other enzymes, as indicated above. Liquiddetergents preferably include stabilised hydrogen peroxide precursors.

It is at present contemplated that, in the detergent composition of theinvention, the peroxidase variant may be added in an amountcorresponding to 0.01-100 mg enzyme per liter of wash liquor.

The present invention is further illustrated in the following exampleswhich are not in any way intended to limit the scope of the invention asclaimed.

EXAMPLE 1 Construction of a Plasmid Expressing the N142S Variant ofCoprinus cinereus Peroxidase

1. Cloning of cDNA Encoding a Coprinus cinereus Peroxidase

Construction of a Probe by PCR

Peroxidase cDNA fragments were prepared by polymerase chain reaction(PCR) using specific oligonucleotide primers (R. K. Saiki et al.,Science 239, 1988, pp. 487-491) constructed on the basis of the aminoacid sequence of the Coprinus macrorhizus peroxidase. PCR was carriedout using the Gene Amp kit and apparatus (available from Perkin ElmerCetus, Norwalk, Conn., USA) in accordance with the manufacturer'sinstructions, with the exception that the reaction was conducted at 28°C. for the first three cycles in order to obtain better hybridisation tothe first strand cDNA (prepared from mRNA obtained from Coprinuscinereus, IFO 8371) and subsequently at 65° C. for 30 cycles of PCR.

The following specific primers were used for PCR:

                  T  T 1. 5′-GCGCGAATTCGTNGGNATNAACCACGG-3′ (SEQ IDNO:3)             A  A 2. 3′-TACAGNTTGACGGGNGGCCTAGGCG-5′ (SEQ ID NO:4)                 A     T  T 3. 5′-GCGAATTCACNCCNCAGGTNTTCGACAC-3′ (SEQID NO:5)           A        T  A 4. 3′-GGNAAGGGNCCNCTCAAGCCTAGGCG-5′(SEQ ID NO:6)                 A 5. 5′-GCGCGAATTCTGGCAGTCNAC-3′ (SEQ IDNO:7)                        A 6. 5′-GCGCGAATTCTGGCAGAGNATG-3′ (SEQ IDNO:8)                T 7. 3′-CGNTACCGNTTCTACAGCCTAGG-5′ (SEQ ID NO:9)

“N” denoting a mixture of all four nucleotides.

The primers were combined as follows: 1 with 2, 3 with 4, 5 with 7, 6with 7, 1 with 4, 1 with 7 and 3 with 7. The PCR fragments were thusextended with an EcoRI site at the 5′-end and a BamHI site at the3′-end. The PCR reactions were analysed on a 1% agarose gel. Bands ofthe expected size were found in all reactions. To verify that the bandscorresponded to peroxidase-specific sequences, the gel was subjected toSouthern blotting and hybridised to an oligonucleotide probe with thefollowing sequence

     T  A  A  A  T 5′-GTCTCGATGTAGAACTG-3′ (SEQ ID NO:10)        T

which is positioned between PCR primers 3 and 4. The probe was found tohybridise to bands of approximately 130 bp, 420 bp, 540 bp and 240 bp,thus confirming that the DNA bands observed correspond to peroxidasesequences.

DNA from the various PCR reactions was digested with EcoRI and BamHI andcloned into the plasmid pUC19 (C. Yanisch-Perron et al., Gene 33, 1985,pp. 103-119). Colonies containing the correct PCR fragments wereidentified by hybridisation using the oligonucleotide probe specifiedabove. DNA from positive colonies was analysed by restriction enzymemapping and partial DNA sequence analysis as described by Sanger et al.,Proc. Natl. Acad. Sci. USA 74, 1977, pp. 5463-5467. A 430 bp fragmentfrom one of the clones, obtained by using primer 1 and 4, was used toscreen a Coprinus cinereus cDNA library as described below.

Construction of a Coprinus cinereus cDNA Library in E. coli

Total RNA was extracted from homogenized Coprinus cinereus (IFO 8371)mycelium, collected at the time for maximum activity of the peroxidaseby methods as described by Boel et al. (EMBO J., 3: 1097-1102, 1984) andChirgwin et al. (Biochemistry (Wash), 18: 5294-5299, 1979).Poly(A)-containing RNA is obtained by two cycles of affinitychromatography on oligo(dT)-cellulose as described by Aviv and Leder(PNAS, USA 69:1408-1412, 1972). cDNA is synthesized by means of a cDNAsynthesis kit from Invitrogen according to the manufacturer'sinstructions. About 50.000 E. coli recombinants from the Coprinuscinereus cDNA library were transferred to Whatman 540 paper filters. Thecolonies were lysed and immobilized as described by Gergen et al.(Nucleic Acids Res. 7, 2115-2135, 1979). The filters were hybridizedwith the ³²P-labelled 430 bp peroxidase-specific probe in 0.2×SSC, 0.1%SDS. Hybridization and washing of the filters was conducted at 65° C.followed by autoradiography for 24 hours with an intensifier screen.After autoradiography, the filters were washed at increasingtemperatures followed by autoradiography for 24 hours with anintensifier screen. In this way, more than 50 positive clones wereidentified. Miniprep plasmid DNA was isolated from hybridizing coloniesby standard procedures (Birnboim and Doly Nucleic Acids Res. 7,1513-1523, 1979), and the DNA sequence of the cDNA insert was determinedby the Sanger dideoxy procedure (Sanger et al., Proc. Natl. Acad. Sci.USA 74, 1977, pp. 5463-5467). The peroxidase cDNA fragment was excisedfrom the vector by cleavage with HindIII/XhoI and was purified byagarose gel electrophoresis, electroeluted and made ready for ligationreactions. The cDNA fragment was ligated to HindIII/XhoI digested pHD414to generate pCiP in which the cDNA is under transcriptional control ofthe TAKA promotor from Asperaillus oryzae and the AMG terminator fromAspergillus niger.

Construction of the Aspergillus Expression Vector pHD414

The vector pHD414 is a derivative of the plasmid p775 (described in EP238 023). In contrast to p775, pHD414 has a string of unique restrictionsites between the promotor and the terminator.

The plasmid was constructed by removal of an approximately 200 bp longfragment (containing undesirable restriction sites) at the 3′ end of theterminator, and subsequent removal of an approximately 250 bp longfragment at the 5′ end of the promotor, also containing undesirablerestriction sites. The 200 bp region was removed from p775 by cleavagewith NarI (positioned in the pUC vector) and XbaI (positioned just 3′ tothe terminator), subsequent filling in the generated ends with KlenowDNA polymerase+dNTP, purification of the vector fragment on gel andreligation of the vector fragment. The DNA was transformed into E. coliMC1061 as described above. 10 colonies (pHD413-1 to -10) were selectedand analyzed by restriction enzyme analysis. One of the clonesexhibiting the expected band pattern in the restriction enzyme analysiswas used in the construction of pHD414.

pHD413 was cut with StuI (positioned in the 5′ end of the promoter) andPvuII (positioned in the pUC vector) and fractionated on a gel. Thevector fragment was purified, religated and transformed into E. coliMC1061. 12 colonies were selected and analyzed by restriction enzymeanalysis. All 12 clones exhibited the expected band pattern. The plasmidpHD414 is shown in FIG. 1.

2. 3-step PCR mutagenesis:

3-step mutagenisation involves the use of four primers:

Mutagenisation primer (=A): 5′-CAG GAG TTC CCA ACC-3′(SEQ ID NO: 11)

PCR Helper 1 (=B): (SEQ ID NO:12) 5′-TGA TCA TAG TAC CAT CTA ATT ACA TCAAGC GGC-3′ PCR Helper 2 (=C): (SEQ ID NO:13) 5′-CTG TAA TAC GAC TCACTA-3′ PCR Handle (=D): (SEQ ID NO:14) 5′-TGA TCA GAC TAG TAC CAT-3′

Primer A and B were diluted to 20 pmol/μl. Primer C and D were dilutedto 100 pmol/μl.

All 3 steps were carried out in a 10×PCR buffer containing: 100 mMTris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl₂, 0.1% gelatin, 200 μl of eachof 2 mM dATP, 2 mM dCTP, 2 mM dGTP, 2 mM TTP, and 200 μl of H₂O.

In step 1, a reaction mixture composed of 10 μl of 10×PCR buffer, 50 μlof 2×nucleotide solution, 5 μl of primer A, 5 μl of primer B, 1 μl ofpCiP (0.05 μg/μl), 30 μl of H₂O, 0.5 μl of Taq polymerase, and 80 μl ofparaffin, was run through 1 cycle consisting of 2 minutes at 94° C., 15cycles consisting of 1 minute at 94° C., 1 minute at 50° C. and 2minutes at 72° C., 15 cycles consisting of 1 minute at 94° C., 1 minuteat 50° C. and 3 minutes at 72° C., and 1 cycle consisting of 5 minutesat 72° C.

10 μl of the PCR product was purified on an agarose gel and redissolvedin 10 μl of H₂O. Then, step 2 was carried out. A reaction mixturecomposed of 10 μl of 10×PCR buffer, 50 μl of 2×nucleotide solution, 5 μlof the purified product of step 1, 1 μl pCiP (0.05 μg/μl), 30 μl of H₂O,0.5 μl of Taq polymerase, and 80 μl of paraffin, was run through 1 cycleconsisting of 5 minutes at 94° C., 2 minutes at 50° C. and 10 minutes at72° C.

To the step 2 reaction mixture, 1 μl of primer C and 1 μl of primer Dwere added. The PCR reaction was carried out as described for step 1.

3. Isolation of the Mutated Restriction Fragment:

The product from step 3 was isolated from an agarose gel andre-dissolved in 20 μl H₂O. Then, it was digested with the restrictionenzymes XbaI and KpnI in NEBuffer 2 (New England Biolabs) supplementedwith bovine serum albumin (BSA) in a total volume of 20 μl at 37° C.overnight. The 570 bp XbaI/KpnI fragment was isolated from an agarosegel.

4. Ligation to Expression Vector pCiP:

The expression plasmid pCiP was cleaved with XbaI and KpnI under theconditions indicated above and the large fragment was isolated from anagarose gel. To this vector, the mutated fragment isolated above wasligated and the ligation mixture was used to transform E. coli. Thepresence and orientation of the fragment was verified by cleavage of aplasmid preparation from a transformant with restriction enzymes.Sequence analysis was carried out on the double-stranded plasmid usingthe di-deoxy chain termination procedure developed by Sanger. Theresulting plasmid is identical to pCiP except for the altered codon.

EXAMPLE 2 Construction of Plasmids Expressing other Variants of Coprinusperoxidase

The following mutants were constructed using the same method asdescribed in Example 1, except that other restriction enzymes were usedfor digesting the PCR-product and the vector used for recloning themutated fragment. Mutations and primers used for the modifications arelisted below.

Muta- tion Primer A sequence N142T 5′-CAG GAG CAC TAG TTC CCA ACC C-3′(SEQ ID NO:15) D56N 5′-TTT TCC ATA ACG CGA TCG-3′ (SEQ ID NO:16) G156F5′-GGT CCC TTT AAC ACT GTC-3′ (SEQ ID NO:17) N142P 5′-CAG GAG CCC GAGTTC CCA AC-3′ (SEQ ID NO:18) N142D 5′-CAG GAG CGA CTC TTC CCA AC-3′ (SEQID NO:19) G154E+ 5′-GAT CCC CGA GCC CTT CGA AAC TGT (SEQ ID G156F+ CACT-3′ NO:20) N157E G154E+ 5′-GAT CCC CGA GCC CTT TAA CAC TGT (SEQ IDG156F C-3′ NO:21) G156F+ 5′-GGT CCC TTC ACT GTC ACT GC (SEQ ID N157F -3′(SEQ ID NO:22) N157F 5′-TCC CGG ATT TAC TGT CAC TGC-3′ (SEQ ID NO:23)D56T 5′-GTT TTC CAT ACT GCG ATC GG-3′ (SEQ ID NO:24) D56I 5′-GTT TTC CATATT GCG ATC GG-3′ (SEQ ID NO:25) D245I 5′-ATG AGG TCG ATC GCT CTC TT-3′(SEQ ID NO:26) D245T 5′-ATG AGG TCC ACG GCT CTC TT-3′ (SEQ ID NO:27)D245N 5′-ATG AGG TCC AAT GCT CTC TT-3′ (SEQ ID NO:28) C22S 5′-AAC AGCCAG AGC TGC GTC T-3′ (SEQ ID NO:29) C23S 5′-AGC CAG TGC AGC GTC TGGTT-3′ (SEQ ID NO:30) Q1S 5′-GCT CTA CCC TCC GGA CCT GGA-3′ (SEQ IDNO:31) Q1E 5′-GCT CTA CCC GAG GGT CCT GGA-3′ (SEQ ID NO:32) A304 5′-AACAAC TGA GCT CCT GTT-3′ (SEQ ID STOP NO:33) C256S 5′-ACC GCC TCG CGA TGGCAA-3′ (SEQ ID NO:34) G154E 5′-TTG ATC CCC GAG CCC GGA AAC-3′ (SEQ IDNO:35) N157E 5′-GGT CCC GGA GAA ACT GTC ACT-3′ (SEQ ID NO:36) T331A+5′-ATT GCT GCA GCC TCA GGC CCT CTC (SEQ ID S338A CCA GCG CTC GCT CCT-3′NO:37) T331N+ 5′-ATT GCT AAT GCC TCA GGC CCT CTC (SEQ ID S383A CCA GCGCTC GCT CCT-3′ NO:38) S8N 5′-GGG GCA CGT GAC GTT CCC GCC-3′ (SEQ IDNO:39) S263N+ 5′-CAT AAC TTC GCT GCT ATT GGT CAT (SEQ ID N265S -3′NO:40) Q38N 5′-CTT GGA CCC ATT GTA GTT-3′ (SEQ ID NO:41) A304S 5′-AACAGG AGC GCT GTT GTT GGA-3′ (SEQ ID NO:42) P-1+ 5′-CTC GCT CTA ATG GAGGGT CCT-3′ (SEQ ID QIE NO:43)

It should be noted that variants in position 1-29 were digested withBamHI and XbaI in NEBuffer 3 (New England Biolabs) supplemented withBSA, resulting in a 160 bp fragment. Variants in position 30-219 weredigested with XbaI/KpnI in NEBuffer 2 supplemeted with BSA, resulting ina 570 bp fragment. Variants in position 220-277 were digested withKpnI/MscI in NEBuffer 2 suplemented with BSA, resulting in a 170 bpfragment. Variants in position 278-363 were digested

with MscI/XhoI in NEBuffer 2 supplemented with BSA, resulting in a 420bp fragment.

EXAMPLE 3 Expression of Coprinus peroxidase Variants in Aspergillusoryzae

Transformation of Aspergillus oryzae or Aspergillus niger (generalprocedure)

100 ml of YPD medium (Sherman et al., Methods in Yeast Genetics, ColdSpring Harbor Laboratory, 1981) was inoculated with spores of A. oryzaeor A. niger and incubated with shaking at 37° C. overnight. The myceliumwas harvested by filtration through miracloth and washed with 200 ml of0.6 M MgSO₄. The mycelium was suspended in 15 ml of 1.2 M MgSO₄. 10 mMNaH₂PO₄, pH=5.8. The suspension was cooled on ice, and 1 ml of buffercontaining 120 mg of Novozym® 234, batch 1687 was added. After 5 minutes1 ml of 12 mg/ml BSA (Sigma type H25) was added, and incubation withgentle agitation was continued for 1.5-2.5 hours at 37° C. until a largenumber of protoplasts was visible in a sample inspected under themicroscope.

The suspension was filtered through miracloth, the filtrate wastransferred to a sterile tube and overlayered with 5 ml of 0.6 Msorbitol, 100 mM Tris-HCl, pH=7.0. Centrifugation was performed for 15minutes at 100×g, and protoplasts were collected from the top of theMgSO₄ cushion. 2 volumes of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH=7.5.10 mM CaCl₂) were added to the protoplast suspension and the mixture wascentrifuged for 5 minutes at 1000×g. The protoplast pellet wasresuspended in 3 ml of STC and repelleted. This procedure was repeated.Finally the protoplasts were resuspended in 0.2-1 ml of STC.

100 μl of the protoplast suspension was mixed with 5-25 μg of theappropriate DNA in 10 μl of STC. Protoplasts from the A1560 strain of A.oryzae (IFO 4177) were mixed with pToC186 (an A. nidulans amdS genecarrying plasmid). The mixture was left at room temperature for 25minutes. 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl₂ and 10 mMTris-HCl, pH=7.5, were added and carefully mixed (twice) and finally0.85 ml of the same solution was added and carefully mixed. The mixturewas left at room temperature for 25 minutes, spun at 2500×g for 15minutes and the pellet was resuspended in 2 ml of 1.2 M sorbitol. Afteranother sedimentation, the protoplasts were spread on the appropriateplates. Protoplasts from the A1560 strain transformed with pToC186 werespread on minimal plates (Cove Biochem. Biophys. Acta 113 (1966) 51-56)containing 1.0 M sucrose, pH=7.0, 10 mM acetamide as nitrogen source and20 mM CsCl to inhibit background growth. After incubation for 4-7 daysat 37° C. spores were picked, suspended in sterile water and spread forsingle colonies. This procedure was repeated and spores of a singlecolony after the second reisolation were stored as definedtransformants.

Production of Recombinant Coprinus cinereus peroxidase Variants in an A.oryzae Strain

Plasmids containing the appropriate mutations in the peroxidase genewere transformed into A. oryzae A1560 by cotransformation with pToC186containing the amds gene from A. nidulans as described above with amixture of equal amounts of pCiP and pToC186 (approximately 5 μg ofeach). Transformants which were able to use acetamide as their solenitrogen source were reisolated twice. After growth on YPD medium(Sherman et al. 1981) for three days culture supernatants were analysedby a peroxidase activity assay using ABTS (vide below). The besttransformants were selected for further studies.

EXAMPLE 4 Hydrogen Peroxide Stability and Substrate Activity of Coprinusperoxidase Variants of the Invention

Wild-type recombinant Coprinus cinereus peroxidase (rCiP) and thevariants G156F, N157F, G154E+G156F+N157E, G154E+G156F, D245T, and D245N,prepared as described above, were tested for their ability to oxidisethe substrates 2,2′-azino-di-[3-ethylbenzthiazoline-6-sulfonic acid],diammonium salt (ABTS), Methyl Orange (MO), vanillin(3-methoxy-4-hydroxybenzaldehyde) (Van), 2-aminobenzoic acid (AMB),veratryl alcohol (Ver) and Mn⁺⁺. Samples of the tested variants werepure and supplied in elution buffer. The enzyme concentration wasdetermined from A₄₀₅ and ε₄₀₅=109 mM⁻. Enzyme stock dilutions used inassays were stable, leading to reproducible oxidation rates.

Assay conditions were substantially as described by Andersen et al. inBiochemical, Molecular and Physiological Aspects of Plant Peroxidases(J. Lobarzewski, H. Greppin, C. Penel and T. Gaspar, Eds.) 1991, pp.169-175, as outlined in Table 1 below.

TABLE 1 rCiP Assays Conditions Velocity Substrate 25° C. mabs/min MO pH= 7.0 465 nm 215 ± 5 [MO] = 46 μM [H₂O₂] = 200 μM [rCiP] = 49 nM  0 −120 s ABTS pH = 7.0 418 nm 163 ± 1 [ABTS] = 1.7 mM [H₂O₂] = 1.0 mM[rCiP] = 50 pM  0 − 120 s Van pH = 7.0 349 nm 419 ± 10 [Van] = 176 μM[H₂O₂] = 232 μM [rCiP] = 1.1 nM  0 − 80 s AMB pH = 7.0 310 nm  40 ± 2[AMB] = 100 μM [H₂O₂] = 100 μM [rCiP] = 11 nM  0 − 100 s Ver pH = 3.5310 nm  31 ± 5 [Ver] = 130 μM [H₂O₂] = 230 μM [LiP] = 500 nM  0 − 200 sMn²⁺ pH = 5.0 300 nm  21 ± 5 [Mn²⁺] = 120 μM [Lac] = 50 mM [H₂O₂] = 35μM [MnP] = 11 nM 50 − 200 s H₂O₂ pH 7.0 420 nm t_(½) = 14 min [H₂O₂] =4.4 mM [rCiP] = 5.5 μM

Hydrogen peroxide stability was measured as the irreversibleinactivation of CiP-III (formed in excess of hydrogen peroxide, cf. M.B. Andersen et al., Acta Chim. Scand. 45, 1991, pp. 1080-1086). Theabsorbance at 405 nm was measured over time, and the time required todecrease the absorbance at 405 nm to half the original value is definedas the half-life. The test solution was composed of 10 mM sodiumphosphate buffer, pH 7, 5.5 μM Coprinus cinereus peroxidase, 4.4 mM (or800 times molar excess) of hydrogen peroxide, measured at 25° C.

It appears from Table 2 below that none of the peroxidase variantsexhibited increased activity compared to wild-type rCiP. One variantretained wild-type activity, while the other variants exhibiteddecreased activity. The variant G154E+G156F+N157E had a decreasedactivity level by about a factor 2. The active site variants (D245N andD245T) exhibited the most pronounced changes in activity and substratespecificity. The variants G154F and N157E retained wild-type stability,while the variants G154E+G156F+N157E and G154E+G156F had a four timesincreased stability. D245N had a decreased half-life, while D245T had ahalf-life of about an hour.

TABLE 2 rCiP mutants Substrate oxidation, mabs/min Stock solutiont_(1/2) CiP μM MO ABTS Van AMB Ver Mn²⁺ min rCiP 112  215 ± 5 163 ± 1 419 ± 10 40 ± 2 0 0 14 G156F 140  212 ± 2 134 ± 1 437 ± 2 42 ± 1 0 0 20N157F 34 117 ± 0  83 ± 1 343 ± 4 27 ± 1 0 0 18 G154E 233   46 ± 1  38 ±2  39 ± 1 0 0 0 55 G156F G157E G154E 25  25 ± 2  15 ± 1  31 ± 2 0 0 0 55G156F D245T 23  10 ± 0  5 ± 1 0 0 0 0 ˜60   D245N 60  5 ± 0 24 ± 2 0 0 00  6

EXAMPLE 5

Bleaching Effect of a Peroxidase Variant of the Invention

A peroxidase variant of the invention, G154E+G156F+N157E, whichexhibited favourable stability properties in the assay described inExample 4, was tested for its ability to bleach dyes in the presence ofdifferent accelerators. Wild-type rCiP was used as control. The enzymeconcentration was determined from A₄₀₅ and ε₄₀₅=109 mM⁻¹ to be 233 μMfor the variant and 110 μM for the wild-type rCiP.

Wild-type rCiP and the G154E+G156F+N157E variant were assayed for dyebleaching under the following conditions:

The test solution was composed of 10 mM sodium phosphate buffer, pH 8,200 μM hydrogen peroxide, 50 μM oxidisable substrate and 10 nM rCiP orperoxidase variant. The dyes used in the assay were Methyl Orange,Direct Blue 1 and 90 and Acid Red 151. The concentration of dye wasabout 4 μM. The test temperature was 25° C. The oxidisable substrateswere p-hydroxybenzene sulphonate, vanillin, 7-hydroxycoumarin and4-hydroxycinnamic acid, commonly used as bleach accelerators.

The bleaching affect was monitored over about 15 minutes as the changein absorbance measured at 465 nm for Methyl Orange, 600 nm for DirectBlue 1, 610 nm for Direct Blue 90 and 510 nm for Acid Red 151. In thepresence of 7-hydroxycoumarin, Methyl Orange was bleached to the samelevel of absorbance by the wild-type and variant peroxidase. Thepresence of vanillin gave rise to a slight bleaching effect for thevariant, while the other two accelerators did not have any effect onbleaching. In the presence of 7-hydroxycoumarin, Direct Blue 90 wasbleached to the same level of absorbance by the two enzymes, thepresence of vanillin gave rise to a slight bleaching effect for thevariant, and the other two accelerators had no effect. Direct Blue 1 wasbleached completely by the wild-type peroxidase in the presence of allfour accelerators, while the peroxidase variant was able to bleach thedye in the presence of 7-hydroxycoumarin and vanillin, and partly in,the presence of 4-hydroxycinnamic acid, whereas p-hydroxybenzenesulphonate had no effect. In the presence of 7-hydroxycoumaric acid,Acid Red 151 was bleached completely by the peroxidase variant atapproximately the same rate and overall performance as by the wild-typeenzyme, while the other two accelerators had no effect.

Based on these results, it is concluded that the peroxidase variantG154E+G156F+N157E bleaches each of the four dyes as efficiently as thewild-type enzyme in the presence of 7-hydroxycoumarin as accelerator. Inthe presence of vanillin, the variant bleaches two of the dyescompletely. The variant is unable to bleach any of the four dyes in theabsence of accelerator. This may be explained by the location of themutations introduced into the variant. They are located on the proteinsurface near the substrate channel, making it less accessible to largemolecules such as dyes.

EXAMPLE 6

Reactions With ABTS

Purified CiP and variants produced as described in Example 2 were storedin 3 M ammonium sulfate at 4° C. The 3 M ammonium sulfate supernatantwas decanted after centrifugation, and the precipitate was solubilizedin 10 mM CaCl₂ to a protein concentration of 1% (w/w). Rate constants ofCiP(wt) and variants were determined by steady-state measurements usinghydrogen peroxide (Merck) as the oxidizing substrate and ABTS as thereducing substrate. The reactions were monitored with a Beckmann DU70spectrophotometer. The concentration of aqueous stock solutions of ABTSwas determined spectroscopically at 340 nm using a molar absorptivity of36 mM⁻¹cm⁻¹. The concentration of aqueous stock solutions of hydrogenperoxide was determined spectroscopically at 240 nm using a molarabsorptivity of 43.6 M⁻¹cm⁻¹. The reactions were performed in potassiumphosphate buffer at pH 6.8, and the temperature was 25° C. The ionicstrength was kept constant at 0.1 M by regulation of the potassiumphosphate concentration. Reactants were used in the following ranges ofconcentration: Peroxidase (2×10⁻⁹-10⁻¹⁰M), hydrogen peroxide (10-100 μM)and ABTS (1 μM-5 mM). The rate of oxidation of ABTS was monitored at 414nm with a molar absorptivity of 31.1 mM⁻¹cm⁻¹.

ABTS oxidation by peroxidase is described below. Two ABTS molecules areoxidized by an electron transfer to a rather stable radical.

According to Dunford in Peroxidase in Chemistry and Biology, J. Evers etal (eds.), Vol. 2, pp. 2-24, CRC Press, 1991, the rate constant forformation of compound I (k₁) and for the second oxidation of ABTS (k₃)can be determined using the following equation, assuming k₂>>k₃;$\frac{2\lbrack{POD}\rbrack}{v} = {\frac{1}{k_{3}\lbrack{ABTS}\rbrack} + \frac{1}{k_{1}\left\lbrack {H_{2}O_{2}} \right\rbrack}}$

[POD] is the total concentration of peroxidase, v is the initial rate ofthe oxidation of ABTS. At constant [H₂O₂] plots of 2[POD]/v vs.1/[ABTS], 1/k₃ is obtained from the slope and 1/k₁[H₂O₂] from theintercept.

TABLE 3 Rate Constants k₁ (reduction of H₂O₂) and k₃ (oxidation of ABTS)for CiP and CiP variants at pH 6.8 and 25° C. k₁ (μM⁻¹ s⁻¹) % of wt k₁k₃ (μM⁻¹ s⁻¹) % of wt k₃ wild type 7.9 ± 0.2 100  35.0 ± 1.0 100 G154E3.2 ± 0.1 41  0.6 ± 0.1 1.7 G156F 7.1 ± 0.3 90 37.0 ± 2.0 106 N157E 5.4± 0.1 68  7.8 ± 0.1 22 N157F 5.8 ± 0.1 73 26.0 ± 1.0 74 G156F-N157F 5.7± 0.2 72 62.0 ± 2.0 177 G154E-G156F- 3.6 ± 0.3 46  2.6 ± 0.1 7.4 N157E

The rate constant k₁ for formation of compound I at pH 6.8, see Table 3,is 7.9 μM⁻¹ s⁻¹, and k₁ is lower for all mutants, especially the mutantscontaining the G154E substitutions where k₁ is less than half of wildtype. Wild type and G156F have the same magnitude for the rate constantsof ABTS oxidation (k₃), see Table 3, Col. 3, and the N157F mutant has ak₃ value of 26 μM⁻¹s⁻¹, which is 74% compared to wt, but the variantG156F-N157F is almost twice as active as wt.

43 1 1306 DNA Coprinus cinereus 1 actatgaagc tctcgctttt gtccaccttcgctgctgtca tcatcggtgc cctcgctcta 60 ccccagggtc ctggaggagg cgggtcagtcacttgccccg gtggacagtc cacttcgaac 120 agccagtgct gcgtctggtt cgacgttctagacgatcttc agaccaactt ctaccaaggg 180 tccaagtgtg agagccctgt tcgcaagattcttagaattg ttttccatga cgcgatcgga 240 ttttcgccgg cgttgactgc tgctggtcaattcggtggtg gaggagctga tggctccatc 300 attgcgcatt cgaacatcga attggccttcccggctaatg gcggcctcac cgacaccgtc 360 gaagccctcc gcgcggtcgg tatcaaccacggtgtctctt tcggcgatct catccaattc 420 gccactgccg tcggcatgtc caactgccctggctctcccc gacttgagtt cttgacgggc 480 aggagcaaca gttcccaacc ctcccctccttcgttgatcc ccggtcccgg aaacactgtc 540 actgctatct tggatcgtat gggcgatgcaggcttcagcc ctgatgaagt agttgacttg 600 cttgctgcgc atagtttggc ttctcaggagggtttgaact cggccatctt caggtctcct 660 ttggactcga cccctcaagt tttcgatacccagttctaca ttgagacctt gctcaagggt 720 accactcagc ctggcccttc tctcggctttgcagaggagc tctccccctt ccctggcgaa 780 ttccgcatga ggtccgatgc tctcttggctcgcgactccc gaaccgcctg ccgatggcaa 840 tccatgacca gcagcaatga agttatgggccagcgatacc gcgccgccat ggccaagatg 900 tctgttctcg gcttcgacag gaacgccctcaccgattgct ctgacgttat tccttctgct 960 gtgtccaaca acgctgctcc tgttatccctggtggcctta ctgtcgatga tatcgaggtt 1020 tcgtgcccga gcgagccttt ccctgaaattgctaccgcct caggccctct cccctccctc 1080 gctcctgctc cttgatctgg tgaagatggtacatcctgct ctctcatcat ccctcttagc 1140 tatttatcca atctatctac ctatctatgcagtttctgtt ctatcaccac aggaagcaag 1200 aaagaaaaac aacaatgcaa cgtgagcagaaatcagcaaa aaaataaatc agtatactac 1260 agtaatgagg ccagtttgcg tggtgtcagaagtaagtacg actcgg 1306 2 435 PRT Coprinus cinereus VARIANT (1)...(435)Xaa = Any Amino Acid 2 Thr Met Lys Leu Ser Leu Leu Ser Thr Phe Ala AlaVal Ile Ile Gly 1 5 10 15 Ala Leu Ala Leu Pro Gln Gly Pro Gly Gly GlyGly Ser Val Thr Cys 20 25 30 Pro Gly Gly Gln Ser Thr Ser Asn Ser Gln CysCys Val Trp Phe Asp 35 40 45 Val Leu Asp Asp Leu Gln Thr Asn Phe Tyr GlnGly Ser Lys Cys Glu 50 55 60 Ser Pro Val Arg Lys Ile Leu Arg Ile Val PheHis Asp Ala Ile Gly 65 70 75 80 Phe Ser Pro Ala Leu Thr Ala Ala Gly GlnPhe Gly Gly Gly Gly Ala 85 90 95 Asp Gly Ser Ile Ile Ala His Ser Asn IleGlu Leu Ala Phe Pro Ala 100 105 110 Asn Gly Gly Leu Thr Asp Thr Val GluAla Leu Arg Ala Val Gly Ile 115 120 125 Asn His Gly Val Ser Phe Gly AspLeu Ile Gln Phe Ala Thr Ala Val 130 135 140 Gly Met Ser Asn Cys Pro GlySer Pro Arg Leu Glu Phe Leu Thr Gly 145 150 155 160 Arg Ser Asn Ser SerGln Pro Ser Pro Pro Ser Leu Ile Pro Gly Pro 165 170 175 Gly Asn Thr ValThr Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe 180 185 190 Ser Pro AspGlu Val Val Asp Leu Leu Ala Ala His Ser Leu Ala Ser 195 200 205 Gln GluGly Leu Asn Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr 210 215 220 ProGln Val Phe Asp Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys Gly 225 230 235240 Thr Thr Gln Pro Gly Pro Ser Leu Gly Phe Ala Glu Glu Leu Ser Pro 245250 255 Phe Pro Gly Glu Phe Arg Met Arg Ser Asp Ala Leu Leu Ala Arg Asp260 265 270 Ser Arg Thr Ala Cys Arg Trp Gln Ser Met Thr Ser Ser Asn GluVal 275 280 285 Met Gly Gln Arg Tyr Arg Ala Ala Met Ala Lys Met Ser ValLeu Gly 290 295 300 Phe Asp Arg Asn Ala Leu Thr Asp Cys Ser Asp Val IlePro Ser Ala 305 310 315 320 Val Ser Asn Asn Ala Ala Pro Val Ile Pro GlyGly Leu Thr Val Asp 325 330 335 Asp Ile Glu Val Ser Cys Pro Ser Glu ProPhe Pro Glu Ile Ala Thr 340 345 350 Ala Ser Gly Pro Leu Pro Ser Leu AlaPro Ala Pro Xaa Ser Gly Glu 355 360 365 Asp Gly Thr Ser Cys Ser Leu IleIle Pro Leu Ser Tyr Leu Ser Asn 370 375 380 Leu Ser Thr Tyr Leu Cys SerPhe Cys Ser Ile Thr Thr Gly Ser Lys 385 390 395 400 Lys Glu Lys Gln GlnCys Asn Val Ser Arg Asn Gln Gln Lys Asn Lys 405 410 415 Ser Val Tyr TyrSer Asn Glu Ala Ser Leu Arg Gly Val Arg Ser Lys 420 425 430 Tyr Asp Ser435 3 27 DNA Artificial Sequence Primer 3 gcgcgaattc gtnggnatna accacgg27 4 25 DNA Artificial Sequence Primer 4 gcggatccgg ngggcagtwn gacat 255 28 DNA Artificial Sequence Primer 5 gcgaattcac nmcncaggtn ttcgacac 286 26 DNA Artificial Sequence Primer 6 gcggatccga actcnccngg gaangg 26 721 DNA Artificial Sequence Primer 7 gcgcgaattc wggcagtcna c 21 8 22 DNAArtificial Sequence Primer 8 gcgcgaattc tggcagarna tg 22 9 23 DNAArtificial Sequence Primer 9 ggatccgaca tcttngccat ngc 23 10 17 DNAArtificial Sequence Primer 10 gtytyratrt araaytg 17 11 15 DNA ArtificialSequence Primer 11 caggagttcc caacc 15 12 33 DNA Artificial SequencePrimer 12 tgatcatagt accatctaat tacatcaagc ggc 33 13 18 DNA ArtificialSequence Primer 13 ctgtaatacg actcacta 18 14 18 DNA Artificial SequencePrimer 14 tgatcagact agtaccat 18 15 22 DNA Artificial Sequence Primer 15caggagcact agttcccaac cc 22 16 18 DNA Artificial Sequence Primer 16ttttccataa cgcgatcg 18 17 18 DNA Artificial Sequence Primer 17ggtcccttta acactgtc 18 18 20 DNA Artificial Sequence Primer 18caggagcccg agttcccaac 20 19 20 DNA Artificial Sequence Primer 19caggagcgac tcttcccaac 20 20 28 DNA Artificial Sequence Primer 20gatccccgag cccttcgaaa ctgtcact 28 21 25 DNA Artificial Sequence Primer21 gatccccgag ccctttaaca ctgtc 25 22 23 DNA Artificial Sequence Primer22 ggtcccttct tcactgtcac tgc 23 23 21 DNA Artificial Sequence Primer 23tcccggattt actgtcactg c 21 24 20 DNA Artificial Sequence Primer 24gttttccata ctgcgatcgg 20 25 20 DNA Artificial Sequence Primer 25gttttccata ttgcgatcgg 20 26 20 DNA Artificial Sequence Primer 26atgaggtcga tcgctctctt 20 27 20 DNA Artificial Sequence Primer 27atgaggtcca cggctctctt 20 28 20 DNA Artificial Sequence Primer 28atgaggtcca atgctctctt 20 29 19 DNA Artificial Sequence Primer 29aacagccaga gctgcgtct 19 30 20 DNA Artificial Sequence Primer 30agccagtgca gcgtctggtt 20 31 21 DNA Artificial Sequence Primer 31gctctaccct ccggacctgg a 21 32 21 DNA Artificial Sequence Primer 32gctctacccg agggtcctgg a 21 33 18 DNA Artificial Sequence Primer 33aacaactgag ctcctgtt 18 34 18 DNA Artificial Sequence Primer 34accgcctcgc gatggcaa 18 35 21 DNA Artificial Sequence Primer 35ttgatccccg agcccggaaa c 21 36 21 DNA Artificial Sequence Primer 36ggtcccggag aaactgtcac t 21 37 39 DNA Artificial Sequence Primer 37attgctgcag cctcaggccc tctcccagcg ctcgctcct 39 38 39 DNA ArtificialSequence Primer 38 attgctaatg cctcaggccc tctcccagcg ctcgctcct 39 39 21DNA Artificial Sequence Primer 39 ggggcacgtg acgttcccgc c 21 40 24 DNAArtificial Sequence Primer 40 cataacttcg ctgctattgg tcat 24 41 21 DNAArtificial Sequence Primer 41 cttggaccca ttgtagaagt t 21 42 21 DNAArtificial Sequence Primer 42 aacaggagcg ctgttgttgg a 21 43 21 DNAArtificial Sequence Primer 43 ctcgctctaa tggagggtcc t 21

What is claimed is:
 1. A variant of a parent peroxidase, comprising asubstitution of methionine at position 166 with leucine, valine,isoleucine, phenylalanine, or glutamine, wherein the parent peroxidaseis encoded by SEQ ID NO:1.
 2. A bleaching agent comprising theperoxidase variant of claim 1, optionally in the form of a non-dustinggranulate, a liquid, or a protected enzyme.
 3. The bleaching agent ofclaim 2, further comprising one or more substrates for the peroxidasevariant.
 4. The bleaching agent of claim 3, wherein the substrate ishydrogen peroxide or a hydrogen peroxide precursor.
 5. The bleachingagent of claim 4, wherein the substrate is a perborate or percarbonate.6. The bleaching agent of claim 4, wherein the amount of substratecorresponds to a hydrogen peroxide concentration in the wash liquor ofbetween 10 μM and 20 mM.
 7. The bleaching agent of claim 3, furthercomprising an oxidisable substrate selected from the group consisting of7-hydroxycoumarin, vanillin, p-hydroxycinnamic acid, 2,4-dichlorophenol,p-coumaric acid and p-hydroxybenzene sulphonate.
 8. The bleaching agentof claim 7, wherein the amount of oxidisable substrate is between about1 μM and about 1 mM.
 9. A detergent composition comprising theperoxidase variant of claim
 1. 10. The detergent composition of claim 9,further comprising one or more substrates for the peroxidase variant.11. The detergent composition of claim 10, wherein the substrate ishydrogen peroxide or a hydrogen peroxide precursor.
 12. The detergentcomposition of claim 11, wherein the substrate is a perborate orpercarbonate.
 13. The detergent composition of claim 10, whichadditionally comprises an oxidisable substrate selected from the groupconsisting of 7-hydroxycoumarin, vanillin, p-hydroxycinnamic acid,2,4-dichlorophenol, and p-hydroxybenzene sulphonate.
 14. The detergentcomposition of claim 13, wherein the amount of oxidisable substrate isbetween about 1 μM and about 1 mM.